Development of vaginal microbicides to safely prevent sexual transmission of herpes simplex virus (HSV), a major co-factor for HIV acquisition, is a major health priority. We seek to determine whether RNA interference (RNAi) can be harnessed to prevent transmission and acquisition of HSV and provide a novel strategy for microbicide development. We hypothesize that short interfering double-or single-stranded RNA (siRNA) can be specifically targeted to the vaginal mucosa to decrease expression of cellular and/or viral proteins critical for the establishment of HSV infection. The first priority will be to target cellular proteins required for viral entry. We hypothesize that gene silencing of either the HSV co-receptor, nectin-1, which has been demonstrated to be the major co-receptor for HSV infection of vaginal tissue, and/or focal adhesion kinase (FAK), activation of which plays a pivotal role in entry and nuclear transport of HSV capsids, will inhibit HSV infection. In parallel studies, we will also target candidate viral genes that encode for proteins essential for viral replication, cell-cell spread and immunomodulatory genes. The choice of viral targets will build from experiences gained from studies of HSV deletion viruses that have been evaluated for gene therapy or as vaccine candidates. Enthusiasm for this approach derives from preliminary results obtained for HSV and HIV. A single mucosal application of liposomal complexes of siRNA targeting nectin-1 (HSV) or CCR5 (HIV) decreases gene expression. Transfection of human cervical cells with siRNA targeting FAK substantially inhibits HSV infection. We propose to translate these findings to mucosal models of genital herpes. This NIH Multi-project Cooperative Agreement will apply an iterative approach toward identifying the best siRNA target(s), optimal modification of siRNA, and formulations to prevent genital herpes. In parallel studies, the mucosal response to siRNA application will be evaluated in human cell cultures and in a murine model as it is critical that topical therapy not interfere with innate genital tract defenses. The results obtained may provide compelling support for advancing siRNAs to clinical trials.